Granular Convoluted Tubule Cells Research Articles

An unusual sexually dimorphic mosaic distribution of a subset of kallikreins in the granular convoluted tubule of the mouse submandibular gland detected by an antibody with restricted immunoreactivity.

The granular convoluted tubule of the mouse submandibular gland contains a wide variety of biologically active proteins, including several kallikreins. The tubule is under multihormonal regulation, and is sexually dimorphic, being larger in males than in females. Correspondingly, levels of its various protein secretory products are more abundant in males than in females. However, isoelectric focussing studies show that the true tissue kallikrein, mK1, is more abundant in the female than in the male submandibular gland. In this study, an antiserum was prepared with restricted immunoreactivity for mouse mK1, and possibly other kallikrein family members of low abundance in the mouse submandibular gland, and used for the immunocytochemical staining of the granular convoluted tubule cells in the submandibular gland of adult male and female mice, by indirect enzyme-labeled and immunogold-labeled antibody methods for light and electron microscopy, respectively. The distribution of immunoreactive tubule cells showed an unusual sexual dimorphism. In males only a few scattered slender tubule cells were strongly stained, while the more typical large tubule cells were only occasionally weakly positive, and many of them were not stained. By contrast, in females slender tubule cells were not seen, and about two thirds of the more typical tubule cells showed moderate to strong immunostaining. Immunoelectron microscopy revealed that immunostaining was confined to the secretion granules in granular convoluted tubule cells in both sexes. The slender tubule cells of males had many strongly stained small apical secretion granules and occasional basal infoldings; in the weakly positive larger more typical tubule cells not all secretion granules were positive, and there was intergranular variation in the intensity of staining of positive granules. In females, although more tubule cells were stained, intergranular variations in staining intensity were also noted. In both sexes, many tubule cells did not contain any secretion granules that showed immunogold labeling for kallikreins. These findings establish that, in contrast to the situation for the majority of granular convoluted tubules proteins, mK1 and possibly other minor kallikrein family members are more abundant in the granular convoluted tubules of female mice, and that there is considerable variation in the content of these kallikreins not only between different tubule cells, but also in individual secretion granules in any given tubule cell in either sex.

Postnatal changes of gene expression for tissue inhibitors of metalloproteinase-1 and -2 and cystatins S and C, in rat submandibular gland demonstrated by quantitative reverse transcription-polymerase chain reaction

The rat submandibular gland is not fully developed at birth and definitive differentiation takes place postnatally. The steady-state mRNA expression for the four proteinase inhibitor molecules, tissue inhibitors of metalloproteinase (TIMP)-1 and -2, and cystatins S and C, and for a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (G3PDH), in rat submandibular glands was measured by quantitative competitive reverse transcription-polymerase chain reaction (RT-PCR) at different stages of postnatal development. The gene-expression patterns of TIMP-1 and -2 relative to G3PDH were similar to each other. The TIMP-2 and cystatin C genes were more highly expressed than those of TIMP-1 and cystatin S at all stages. Moreover, the gene expressions of TIMP-1 and -2, and of cystatins S and C, were predominant between 1 and 7, and 7 and 12 weeks of age, respectively, and coincided developmentally with the regression of terminal tubule cells and the differentiation of granular convoluted tubule cells, respectively. Quantitative competitive RT-PCR allowed accurate measurement of small changes in the steady-state concentrations of these proteinase-inhibitor mRNA molecules.

Transient appearance of substance P-like immunoreactivity in the granular convoluted tubules of the male mouse submandibular gland: light- and electron-microscopic studies.

The time of appearance and distribution of substance P (SP)-like immunoreactivity in the granular convoluted tubule cells of the developing male mouse submandibular glands were examined, and the subcellular localization of SP-like immunoreactivity was investiagted by electron microscopy. At 25 days of age, SP-like immunoreactivity was first detected in the supranuclear cytoplasm of the granular convoluted tubule cells, which occurred either singly or in small clusters. At 30 and 35 days of age, granular convoluted tubule cells with SP-like immunoreactivity were more numerous than in the earlier stages, as the volume ratio of the cells increased. Not all granular convoluted tubule cells demonstrated SP-like immunoreactivity. The number of cells with SP-like immunoreactivity decreased at 60 days of age, and these cells had completely disappeared at 90 days of age. Most, but not all, secretory granules in the granular convoluted tubule cells were strongly labeled with gold particles, indicating that the subcellular site of SP-like substance is in the secretory granules within the cells. The findings suggest that the physiological role of the SP-like substance secreted from the GCT cells is restricted to the early postnatal stages, and that it may be involved in the development of the oral mucosa or digestive tract as a trophic factor.

Sialogogue-Related Radioprotection of Salivary Gland Function: The Degranulation Concept Revisited

To investigate whether secretory granules play a role in the radiosensitivity of the salivary glands of rats, parotid acinar cells, submandibular acinar cells and/or submandibular granular convoluted tubule (GCT) cells were degranulated prior to irradiation. Degranulation of GCT cells was obtained by pretreatment with phenylephrine (5 mg/kg, t = -60 min) and methacholine (3.75 mg/kg, t = -120 min). Degranulation of acinar cells was attained by pretreatment with isoproterenol (5 mg/kg, t = -90 min). Combinations of pretreatments were also tested. Irradiation was performed with a single dose of 15 Gy of X rays. Samples of parotid and submandibular/sublingual saliva were collected 4 days prior to and 1, 3, 6, 10 and 30 days after irradiation. Pretreatment with phenylephrine, isoproterenol and methacholine plus phenylephrine resulted in less radiation damage to parotid gland function as indicated by the lag phase and flow rate. Since the pretreatment with phenylephrine and phenylephrine plus methacholine did not degranulate parotid gland acinar cells, the observed protective effect on this gland cannot be explained by the "degranulation concept." Furthermore, salivary gland function was significantly greater 3 days after irradiation as a result of pretreatment with phenylephrine and phenylephrine plus methacholine compared to rats given only radiation. This may indicate recovery from damage rather than a reduced amount of initial damage. The sparing was most obvious for the later effects (6-30 days). Submandibular/sublingual gland function was improved significantly after pretreatment with methacholine plus phenylephrine, although no increase in degranulation of GCT cells was observed compared to pretreatment with phenylephrine alone, again not favoring the degranulation concept. The results indicate that the secretory granules do not play the often-assumed important role in the radiosensitivity of the salivary gland. The mechanism underlying the observed improvement of salivary gland function may involve second messenger-induced increases in proliferation of salivary gland cells resulting in recovery of tissue after the irradiation.

Prohormone convertases in mouse submandibular gland: co-localization of furin and nerve growth factor.

Nerve growth factor (NGF) in mouse submandibular glands (SGs) is generated from a 35-kD precursor by proteolytic enzymes that have yet to be identified. Prohormone convertases (PCs) cleave the NGF precursor in vitro, and in this study we questioned whether PCs could process salivary NGF in vivo. mRNA coding for PC2 (but not PC1) was detected on Northern blots of SG mRNA and also by in situ hybridization within parasympathetic neurons of intralobular ganglia. Northern blot and in situ hybridization analyses also detect mRNA coding for furin. In SGs of male mice, furin mRNA levels are high at birth and remain high throughout development. In glands from female mice, levels decline during postnatal development and are lower in adults than in newborns. Immunocytochemistry detects furin immunoreactivity in pro-acinar and ductal cells of glands from newborn and pubescent mice. In glands of adults, furin immunoreactivity is detectable in acinar cells but highest levels are present in NGF-containing granular convoluted tubule cells. These data, taken together with those from previous studies, suggest that furin is a candidate processing enzyme for NGF in mouse submandibular glands.

The rodent granular convoluted tubule cell--an update.

The cells of the granular convoluted tubule (GCT) of the rodent submandibular gland (SMG) are under complex developmental and multihormonal regulation. Recent findings indicate that GCT cells also synthesize transforming growth factor alpha (TGF-alpha), hepatocyte growth factor, erythroid differentiation factor, endothelin, and insulin-like growth factor, as well as several novel androgen-dependent proteins of unknown function. The GCTs of hypophysectomized mice provide a convenient model to study multihormonal regulation of gene expression. The GCT system of the rodent SMG also is a fruitful model for study of hormone receptors.

Emergence of salivary gland cell lineage diversity suggests a role for androgen-independent epidermal growth factor receptor signaling.

Diversity of cell lineages within glandular organs is generated postnatally by differentiation of committed progenitor cells. Fundamental regulatory aspects of this process are not understood. The mouse submandibular salivary gland (SSG) served as model to assess the role of epidermal growth factor (EGF) receptor signaling during emergence of cell lineage diversity. Temporal fluctuations in EGF receptor mRNA levels coincident with crucial differentiative cell lineage transitions were revealed by RNase protection analyses. Between days 2 and 5, when proacinar cells are maturing and striated duct cells emerge, EGF receptor mRNA levels were highest and all differentiating cells exhibited EGF receptor immunoreactivity. EGF receptor mRNA levels then declined sharply and immunoreactivity became confined to ductal cells. At day 11 in male mice, and days 11 and 16 in females, a second increase in EGF receptor mRNA was detected coincident with emergence of granular convoluted tubule (GCT) cells. With completion of androgen-dependent GCT cell differentiation at the onset of puberty, EGF receptor mRNA levels and intensity of immunoreactivity decreased. Androgen effects on EGF receptor mRNA or immunoreactivity could not be detected. These temporally distinct patterns of EGF receptor expression suggest that this signaling pathway is a mechanism of potential importance in emergence of cell lineage diversity in a glandular organ.

Degranulation of rat salivary glands following treatment with receptor-selective agonists.

1. The aim of this study was to find a drug that induces an almost complete degranulation of secretory cells in rat parotid and submandibular glands. 2. Phenylephrine (alpha-adrenergic), isoproterenol (beta-adrenergic) and mecholine (muscarinic cholinergic) were tested. Time and degree of maximal depletion of acinar and granular convoluted tubule cells were determined morphologically. 3. Following phenylephrine-injection (5 mg/kg or 10.2 mg/kg, i.p.), no effect on the acinar granulation level was observed in either of the glands, while about 50-60% granular convoluted tubules were degranulated for at least 120-180 min post-injection. 4. With isoproterenol (5, 10, 40, 70 or 100 mg/kg, i.p.), degranulation of 100% of the acinar cells in the parotid and 80% of the acinar cells in the submandibular gland was observed 90 min post-injection. Granular convoluted tubule cells did not respond to this beta-adrenergic drug. 5. Mecholine (3.75 or 7.5 mg/kg, i.p.) induced mainly degranulation of granular convoluted tubule cells (about 50% after 120 min). Numbers of granulated acinar cells decreased only slightly in both glands (about 10%, 90-120 min). 6. From this study it appears that with a relatively low dosage (5 mg/kg, i.p.) of isoproterenol, a high level of degranulation can be induced in acinar cells of rat parotid and submandibular glands without toxic side effects. Concerning granular convoluted tubules, only moderate degranulation was observed with phenylephrine and mecholine, respectively.

Cell Proliferation Activity in Duct-Ligated Submandibular Gland.

Cellular proliferation activity in duct-ligated submandibular glands (SMGs) of rats were evaluated by using index of proliferating cell nuclear antigen (PCNA) immunostaining and 3H-thymidine incorporation. The normal SMGs showed a very low index (<0.1%) of PCNA immunostaining and 3H-thymidine incorporation in the acinar and all the ductal segments. On day 2 and 3 of duct ligation, index of PCNA staining and 3H-thymidine incorporation increased and reached a peak in degranulated granular convoluted tubules (GCTs), duct like structures (DLSs) and dilated excretory ducts (EDs). On day 5 to 10, the glands showed fibrous proliferation around the DLS, and cells in DLSs and dilated EDs had decreasing index of PCNA staining and 3H-thymidine incorporation. On day 14 and 21, the PCNA staining and 3H-thymidine incorporation had decreased and was mainly confined to the DLSs and basal cells of keratinizing epithelial masses. In the unligated SMG of the opposite side of the duct-ligated animals, proliferating cells were found mainly in the acinar compartment from the 2nd to 5th day of ligation.These results suggested that in case of acceleration of secretry function of SMG, secretory cells may proliferate as for the functional adaptation. However, in the case of obstructive lesions of SMG, GCT cells and ductal basal cells may have more potential for proliferation and these cells may be the major participant cells during glandular regeneration and possibly in the histogenesis of the majority of salivary gland tumors, if not all.

Radiation-Induced Cell Proliferation in the Parotid and Submandibular Glands of the Rat

Repopulation of tissues with cells at damaged sites is an important feature in the recovery of radiation-induced tissue injury. To obtain insight into the regenerative process in salivary gland tissue, proliferative activity was measured as a function of time in the different epithelial cell compartments of rat parotid and submandibular glands after local X irradiation with a single dose of 15 Gy. Bromodeoxyuridine-labeling indices were determined before and 10 h and 1, 3, 6, 10, 15, 20 and 30 days after irradiation. In both glands, X irradiation caused cell death and cell cycle delay manifested during the first day. Three days after irradiation, cell proliferation started in the intercalated duct. Six days after irradiation, proliferation was also observed in acinar and granular convoluted tubule cells. The striated ducts showed proliferative activity starting at day 6 (parotid) and day 10 (submandibular), respectively. The results of this study suggest that after 15 Gy of X rays repopulation takes place in all cell compartments. From the present study it cannot deduce if these cells are originating solely from progenitor cells residing in the intercalated duct or if cells of the other compartments are also stimulated. Proliferative activity was found to be higher in the intercalated duct compartment of the parotid gland than of the submandibular gland, which may be related to the suggested greater radiosensitivity and thus a greater demand for cell replenishment in the parotid gland.

Expression and Localization of Hepatocyte Growth Factor in Rat Submandibular Gland

By combination of in situ hybridization and immunohistochemical techniques, the expression of hepatocyte growth factor (HGF) was demonstrated in the submandibular gland of rats. Both the mRNA signal and immunoreactivity for HGF were localized exclusively to the epithelial cells of granular convoluted tubules, whereas they were absent from the other components of the submandibular gland. In the granular convoluted tubule cells, HGF-immunoreactivity was localized to the apical secretory granules, which was further substantiated by immunoelectron microscopy. These results added HGF to the list of many growth factors that are produced in the rat submandibular gland and secreted into the saliva.

The granular convoluted tubule (GCT) cell of rodent submandibular glands.

The granular convoluted tubule (GCT) is a segment of the duct system of all rodents, situated between the striated and intercalated ducts. It has the peculiar property of synthesizing a large variety of biologically active polypeptides whose role in saliva remains unknown. The literature on the fine structure of GCT cells is critically reviewed. Some recent developments on endocrine regulation of the structure and contents of rodent GCT cells are summarized, with emphasis on EGF, NGF, renin, and kallikrein proteases. A survey of the distribution of GCT cells in several vertebrate families is presented.

Chronotoxicological effect of methyl mercury in rat submandibular gland. Immunohistochemical changes of r-EGF, S-100 protein and keratin.

The effect of toxicity of methyl mercury was investigated in the submandibular gland (SMG) of adult male rats subjected to 12 h light dark cycle (6 a.m. to 6 p.m. light/resting phase; 6 a.m. to 6 p.m. dark/active phase). Two groups of rats received a defined dose of methyl mercury hydroxide at seven different time points during the active (dark) and resting (light) phase over a 24 h period. After 10 d, the rats were killed at 9 a.m. and 9 p.m. in the resting and active phase, respectively. The immunohistochemical distribution pattern of epidermal growth factor (r-EGF), S-100 protein and K8.12 keratin were studied in granular convoluted tubules (GCT). Those rats which received injections during the active phase (6 p.m. to 6 a.m.), showed stronger reactivity for r-EGF; however, the reactivity for S-100 protein was unchanged. On the contrary, in both groups, GCT cells showed intense staining for K8.12 keratin. It is concluded that the detoxification mechanism of mercury appears to be dependent on the chronobiological oscillation pattern of the GCT and their substructures.

Expression, localization and developmental regulation of insulin-like growth factor I mRNA in rat submandibular gland

The mRNA for insulin-like growth factor I (IGF-I) in the submandibular gland of mature and developing rats was examined by Northern blotting and in situ hybridization with an oligonucleotide probe. In the mature adult rat, IGF-I mRNA was expressed at a higher level in the submandibular gland than in the liver, and was localized primarily in the granular convoluted tubule (GCT) cells. A 4.7-kb mRNA on Northern blots, which was expressed only slightly in the liver, proved to be the predominant size species of IGF-I transcripts in the GCT cells, and its level increased progressively with the postnatal development of GCTs in the gland. In addition, a 1.8-kb mRNA for IGF-I was also expressed at a much lower level throughout the acinar and duct systems, irrespective of age. These results have shed a light on the status of IGF-I as one of the many biologically active polypeptides that are produced in the rodent submandibular gland.

Nerve growth factor and epidermal growth factor in mouse submandibular glands: identical diurnal changes and rates of secretagogue-induced synthesis.

The granular convoluted tubule cells of the mouse submandibular gland contain high concentrations of nerve growth factor (NGF) and epidermal growth factor (EGF). Levels of the two growth factors increase in parallel during development and after stimulation by thyroid hormone, androgens, and glucocorticoids, suggesting that similar controls regulate the production of both proteins. At the same time, however, the granular convoluted tubules hypertrophy, with resulting increases in glandular size and protein content. In this study, we have monitored changes in salivary concentrations of NGF and EGF in the absence of detectable changes in submandibular size or protein concentration. Both growth factors exhibited identical diurnal changes, peaking between 1200-2000 h. In addition, after secretagogue-stimulated release of the proteins into saliva, reaccumulation began after 2 h, with normal levels of both molecules attained 6 h posttreatment. These data indicate that submandibular NGF and EGF can be controlled by similar cellular or molecular mechanisms independent of the regulation of generalized growth responses.

Secretory and synthetic changes in the secretory proteinases of mouse submandibular glands after methoxamine stimulation, assessed histochemically by means of D-Val-Leu-Arg-methoxy-naphthylamide.

The secretion of proteinases and their resynthesis in the granular convoluted tubule (GCT) cells of mouse submandibular glands were studied after in vivo methoxamine (Methox) treatment (15mg/kg, i. p.), using a histochemical reaction with D-Val-Leu-Arg-4-methoxy-2-naphthylamide. The amidase reaction product was confined to the secretory granules in GCT cells, in unstimulated and stimulated glands. Methox caused extensive secretion of the granules. An increased staining reaction by the proteinases seemed to occur in granules prior to secretion, and fusions had often taken place. Reaccumulation of proteinases in the newly forming granules was found to start between 6 to 12hr after injection and the process was still incomplete at 48hr, but the granules showed less staining than at the earlier times. The variations observed in staining intensity may relate to the extent of binding with other proteins, and an unbinding of proteinases appears to be a possibility prior to the secretion of granules.

Secretion of glandular kallikrein and renin from the basolateral pole of mouse submandibular duct cells: an immunocytochemical study.

Glandular kallikrein from salivary glands in rats has been measured in the circulation and has been shown to have local vasoactive effects. In mice, renin and epidermal growth factor from the submandibular gland (SM) also reach the circulation, as both have been measured in plasma. The route by which these peptides enter the blood from their site of synthesis in ducts of the SM is unclear. We have investigated by immunocytochemistry the secretory pathways for kallikrein and renin from salivary duct cells in mice. The renal/pancreatic kallikrein-secreting cells of the striated and excretory ducts of the SM were distinguished from the granular convoluted tubule (GCT) cells which secrete other glandular kallikreins on the basis of data obtained in previous studies, in which we used gene-specific oligonucleotide probes to identify the expressing cell types. Renal/pancreatic kallikrein was apparently secreted constitutively from the basolateral surface of striated duct cells and in secretory vesicles from excretory duct cells, whereas apical secretion occurred via the regulated pathway in both cell types. Glandular kallikreins and renin synthesized in GCT cells were secreted from the basolateral surface by dissolution of granules at the cell membrane. There were fenestrated capillaries underlying the duct tree which would enable the secreted products to reach the circulation.

Changes of Calmodulin, S-100 Protein .ALPHA./.BETA. and Keratin in Duct-Ligated Salivary Glands of Rats. An Immunohistochemical Study.

Immunohistochemical study of expression of calmodulin, S-100 α, β protein and cytokeratin K8.12 was carried out on normal and duct-ligated submandibular (SMG) and sublingual glands (SLG) of rats. The normal rat SMG showed calmodulin and S-100 α immunoreactivity in the pillar and transition cells of the granular convoluted tubule (GCT) and striated duct cells. S-100 β staining was positive in acinar cells of SMG as fine granular deposition. A trace immunoreaction product of keratin K8.12 was observed in GCT segments and striated ducts. Three days following duct ligation, immunostaining for calmodulin and S-100 β disappeared in both SMG and SLG, however, S-100 α staining persisted in pillar, transition and striated duct cells. The altered GCT cells of SMG and duct-like structures of SLG in the duct-ligated glands also showed S-100 α staining. In addition, K8.12 immunoreactivity was noted in the duct-like structures, particularly in those cells located luminally. The day 5 duct-ligated specimen of SMG retained positive S-100 α protein in the newly formed duct-like structures and the day 7, 10 and 14 specimens showed squamous metaplasia with positive K8.12 staining at the luminal side of the ducts. Myoepithelial cells in duct-ligated SLG stained positively for K8.12. It is concluded that loss of calmodulin from the ductal cells of SMG and SLG, and S-100 β from acini of SMG marks the earliest and predominant pathological alteration followed by increased proliferation of duct-like structures with cells containing cytokeratin K8.12 as pathological alteration of duct ligation of the salivary glands.

Immunocytochemical localization of rat intestinal 15 kDa protein, a member of cytoplasmic fatty acid-binding proteins.

Rat intestinal 15 kDa protein (I-15P) is a member of the family of cytoplasmic fatty acid-binding proteins. Using a specific antiserum against I-15P, we studied the tissue distribution and subcellular localization of this protein in the entire rat body. By immunoblot analysis of cytosolic proteins, I-15P was detected not only in the distal portion of small intestine but also in the ovary and adrenal gland. Immunohistochemically, I-15P was localized to the absorptive epithelial cells as well as a subpopulation of enterochromaffin cells in the intestine, the lutein cells in the ovary, and subpopulations of cortical cells in the adrenal gland. Furthermore, I-15P-like immunoreactivity was also demonstrated in the surface mucous cells of stomach and the granular convoluted tubule cells of submandibular gland. Immuno-electron microscopy showed that the immunoreactivity was confined to the cytoplasmic matrix region, except in the enterochromaffin cells and granular convoluted tubule cells, where it was localized in the secretory granules. The present findings suggest that I-15P plays a role in the cellular metabolism of steroids.

Expression of peptidylglycine alpha-amidating monooxygenase: an in situ hybridization and immunocytochemical study.

Peptide alpha-amidation, an essential posttranslational modification that confers bioactivity to many neuroendocrine peptides, is catalyzed by peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3). To complement our previous studies on the distribution of PAM in neuroendocrine organs, we have examined expression of the PAM gene in several endocrine tissues by in situ hybridization and immunocytochemistry. In all instances, the autoradiographic densities for PAM mRNA correlated with staining patterns for PAM immunoreactivity. Very high levels of PAM mRNA were found in all heart atrial cardiomyocytes, while much lower levels were present in ventricular cells. In the sublingual gland, PAM was expressed diffusely in both acinar and tubule cells. In contrast, expression of PAM was confined to granular convoluted tubule cells in the submaxillary gland. PAM was expressed at high levels in a subset of adrenal medullary chromaffin cells, and low levels of PAM mRNA and immunoreactivity were also detected in the adrenal cortex. PAM was found predominantly in the calcitonin-producing parafollicular C-cells in the thyroid gland and in the glucagon-containing A-cells in the endocrine pancreas. Collecting and distal tubule cells of the kidney expressed both PAM mRNA and immunoreactivity. The basal cells in testicular seminiferous tubules containing PAM may represent developing germ and Sertoli cells. The cellular localization of PAM within the thyroid gland, adrenal gland, testis, and pancreas correlated with known peptidergic systems, and some of the observed cellular heterogeneity in PAM mRNA expression and immunoreactivity may reflect differences in the levels of amidated peptide production. The expression of PAM in cells not known to produce high levels of alpha-amidated peptides may indicate the production of yet unidentified alpha-amidated bioactive peptides or alternative functions of the PAM protein.